Topoisomerases are ubiquitous enzymes that alter the linking number of DNA. As such, they are likely to be involved in all aspects of DNA metabolism. Their importance is underscored by the fact that in eukaryotes they are the target of potent anti-cancer agents, whereas in prokaryotes, both DNA gyrase and topoisomerase IV (Topo IV), which was purified and characterized during the previous grant period, are targets of the most potent broad-spectrum antibiotics known (e.g., ciprofloxacin). Thus, it is crucial to understand their mechanism of action and the roles they play during DNA metabolism. During the previous grant period, using pBR322 and oriC DNA replication reconstituted with purified proteins, we have shown that the dour E. coli topoisomerases, Topo I, Topo II (DNA gyrase), Topo III, and Topo IV are distinct in their ability to support the various stages of theta-type DNA replication. During the new grant period, we will investigate the biological, biochemical, and structural basis for the differences in topoisomerase action. We have shown that Topos II-IV can all support nascent chain elongation in vitro. Evidence indicating that gyrase is the only enzyme involved in this step in vivo is equivocal. To determine the contributions of Topos III and IV in supporting this step in vivo, we will construct strains carrying various combinations of mutations in these enzymes and determine the effect on DNA synthesis. We have shown that Topo III, a type I topoisomerase that requires a single-stranded DNA-binding site, supports nascent chain elongation. This suggests, because no single-strands exist ahead of the forks, that Topo III acts behind the forks. We also know that both Topos III and IV act more efficiently at this stage if they are present during initiation, suggesting that a preferential binding site is formed at the origin. We will determine whether such binding sites exist and investigate how the excess positive windings formed during theta-type DNA replication are distributed over the template and how this distribution affects the action of the topoisomerases. Topo IV and gyrase show high homology, yet during replication only Topo IV, not gyrase, can support processing of the late intermediate and decatenation of the daughter molecules. On the other hand, only gyrase, not Tops IV, and supercoil DNA. We will investigate the structural basis for these differences by constructing chimeric Topo IV-gyrase molecules and assessing the associated activities and by solving the crystal structure of Topo IV. Topo IV acts subsequent to termination of replication and prior to partition. Thus, it is likely to be involved in a signal transduction pathway that connects DNA replication and cell division. To detect other proximal gene products involved in this pathway, we will isolate high- copy suppressors of dominant-lethal Topo IV mutations.